This invention is directed to monoclonal antibodies and Fv specifically binding CD2 antigen and the use of such monoclonal antibodies in preventing the production of the human immunodeficiency virus (HIV-1) in HIV-1-infected T cells.
Acquired immunodeficiency syndrome (AIDS) has become one of the most feared diseases of the latter part of the twentieth century. In the United States alone, about 40,000 new cases of AIDS are diagnosed yearly and at least 200,000 cases have occurred since the disease became recognized about 1981. It is believed that from 1 million to 1.5 million Americans are infected with HIV-1, which means that they are at risk to develop AIDS and are likely to do so unless a means can be found for preventing the development of the full-blown illness in patients infected with the virus. Worldwide, the number of cases of AIDS runs into the millions, with certain areas of Africa and the Caribbean having the highest rates of infection.
Until now, although the amount of money spent for research on AIDS equals or exceeds that spent for research on diseases such as cancer and heart disease, no cure is yet available and the disease is considered uniformly fatal. Although several treatments have been developed that appear to slow the progression of the disease, at least in some patients, such as the drugs azidothymidine (AZT) and dideoxyinosine (DDI), these treatments are regarded only as palliatives. They do not work in all patients, and their effectiveness tends to decrease over the course of the disease. Thus, additionally treatments for AIDS are urgently needed.
It is generally believed that infection with the human immunodeficiency virus (HIC-1) is required for development of AIDS, although other factors may contribute to the establishment and the progress of the disease, because the disease is often associated with drug abuse and with exposure to other pathogens that may contribute to or enhance the immunosuppressive effects. HIV-1 is a retrovirus of the lentivirus subfamily. Like other retroviruses, its life cycle involves reverse transcription and incorporation of the viral RNA genome as DNA into the genome of the infected T cell. Activation of the virus results in transcription and translation, leading to production of the virus and depletion of CD4+ T cells.
One of the characteristics of AIDS is the presence of opportunistic infections. These infections include diarrhea caused by several bacterial species, tuberculosis caused by avian and bovine mycobacteria, viral infections such as cytomegalovirus infection and shingles caused by herpesvirus, fungal infections such as candidiasis, aspergillosis, and histoplasmosis, and protozoan infections such as toxoplasmosis and pneumocystis pneumonia. These infections are frequently the actual cause of death in patients with AIDS, and case much morbidity even when not fatal. Although treatments exist for a number of these opportunistic infections, such as aerosolized pentamidine for pneumocystis pneumonia, these treatments are often unsuccessful because of the general health of the patients and their decreased resistance to infection brought about by AIDS itself.
Thus, improved methods of treating such infections are required.
Like other lentiviruses, HIV-1 can remain latent within the DNA of infected CD4+ T lymphocytes, often for long periods. This latency is poorly understood, but is likely dependent on the state of activation of the host CD4+ T cell (Z. F. Rosenberg and A. F. Fauci, xe2x80x9cMinireview: Induction of Expression of HIV in Latently or Chronically Infected Cells,xe2x80x9d AIDS Res. Hum. Retro, 5: 1-4 (1989)).
Activation of T cells is dependent on a complex scheme of interactions between them and other cells designated as antigen presenting cells (ACs). These interactions are mediated by specific binding between molecules on the cell surface of T cells and molecules on the surfaces of APCs. One such interaction is between CS2 on the surface of T cells and its ligand LFA-3 on APC. The CD2 molecule, when crosslinked by anti-CD2 antibodies, is capable of directly activating T cells. The LFA-3 ligand is also referred to as a counter-receptor. Other receptor/counter-receptor pairs are ICAM-1/CD18 and CS28/B7.
One of the paradoxes that has complicated both the effort to understand AIDS and the effort to treat the opportunistic infections associated with it is that attempts by HIV-1-infected lymphocytes to respond to organisms responsible for opportunistic infections results in increased production of HIV-1 virus by infected cells. This is shown by results indicating that response to staphylococcal enterotoxin (xe2x80x9csuperantigenxe2x80x9d or xe2x80x9cSAgxe2x80x9d) increases HIV-1 production from infected cells in vitro.
Much remains to be understood about activation of HIV-1 production in infected lymphocytes. Various stimuli that have been shown to induce HIV-1 production by infected T-cell lines include the following: (1) T-cell antigens; (2) mitogens; (3) various cytokines such as TNFxcex1, IL-1, and IL-6; and (4) other viruses (Rosenberg and Fauci (1989), supra; Z. F. Rosenberg and A. F. Fauci, xe2x80x9cActivation of Latent HIV Infection,xe2x80x9d J. NIH Res. 2:41-45 (1990)).
However, relatively little is known about mechanisms that activate HIV-1 expression in CD4+ T cells that contain integrated HIV-1 provirus.
Accordingly, there is a need for a method of suppression or blocking of HIV-1 production without markedly altering immunological functions and immune response to pathogens other than HIV-1, or, if some response is suppressed in order to prevent HIV-1 production, to do so without depressing the clinical status of the patient. Such a method would be useful in treating opportunistic infections and in extending the life of patients infected with HIV-1.
We have developed a chimeric humanized single-chain recombinant antibody, CD2 SFv-Ig, specifically binding to CD2 antigen, that can be used to suppress HIV-1 virus production in HIV-1-infected T cells, as well as other antibodies specifically binding CD2 antigen. We have also developed methods for suppressing virus production and treating subjects infected with HIV-1 using CD2 SFv-Ig or other antibodies specifically binding antigens involved in the intercellular interaction between T lymphocytes and monocytes.
Antibodies according to the present invention includes a single-chain recombinant antibody selected from the group consisting of:
(1) chimeric single-chain recombinant antibody CD2 SFv-Ig produced by expression of the construct cloned in recombinant Escherichia coli culture ATCC No. 69277;
(2) a antibody having complementarity-determining regions identical with those of CD2 SFv-Ig; and
(3) a antibody competing with CD2 SFv-Ig for binding to CD2 antigen at least about 80% as effectively on a molar basis as CD2 SFv-Ig.
Preferably, the antibody completes at least about 90% as effectively on a molar basis as CD2 SFv-Ig for binding to CD2 antigen.
The complementarity-determining regions of either H chain origin or L chain origin can be identical in sequence to those of cD2 SFv-Ig.
Preferably, the antibody is chimeric single-chain recombinant antibody CD2 SFv-Ig.
Another aspect of the invention is an antibody modified from the antibody described above by deletion of at least a portion of the Ig-derived amino acid sequence therefrom. The entire Ig-derived amino acid sequence can be deleted. Such an antibody can be derived from CD2 SFv-Ig.
The antibody can be labeled with a detectable marker. The detectable marker can be selected from the group consisting of enzymes, paramagnetic materials, members of the avidin-biotin specific binding pair, fluorophores, chromophores, chemiluminophores, heavy metals, and radioisotopes.
Alternatively, the antibody can be conjugated to a therapeutic agent. The therapeutic agent can be selected from the group consisting of antinenoplastic agents, lymphokines, and toxins. Suitable lymphokines include interleukins, interferons, and tumor necrosis factors, in particular, interleukin-2. Suitable toxins include ricin, pseudomonas exotoxin, and diphtheria toxin.
Another aspect of the invention is a pharmaceutical composition comprising:
(1) a chimeric humanized single-chain recombinant anti-CD2 antibody according to the present invention in a quantity sufficient to inhibit production of HIV-1 virus in infected T cells in a patient infected with HIV-1; and
(2) a pharmaceutically acceptable carrier.
Another aspect of the invention is a recombinant Escherichia coli cell stably transformed by a construct capable of expressing CD2 SFv-Ig chimeric humanized single-chain recombinant antibody in mammalian cells and deposited with the American Type Culture Commission as ATCC No. 69277.
Yet another aspect of the invention is a DNA construct capable of expressing CD2 SFv-Ig chimeric humanized single-chain recombinant antibody in mammalian cells and including murine complementarity-determining regions and human constant regions.
Yet another aspect of the invention is a method for inhibiting the production of HIV-1 in HIV-1-infected T cells. The method comprises the steps of:
(1) selecting T cells infected with HIV; and
(2) contacting the infected T cells with a antibody capable of disrupting cell-surface interactions between CD4+ T lymphocytes and monocytes in order to inhibit the production of HIV-1 in the infected T cells, the cells being contacted with the antibody in a quantity sufficient to inhibit the production of HIV-1 in the contacted T cells.
Preferably, the antibody is specific for CD2 and blocks the binding with CD2 antigen with its counter-receptor LFA-3. More preferably, the antibody is a antibody according to the present invention as described above; most preferably, the antibody is CD2 SFv-Ig. The antibody can also be a monclonal antibody to CD18, a monoclonal antibody to counter-receptor LFA-3, or a monoclonal antibody to counter-receptor ICAM-1. The antibody can be conjugated to a therapeutic agent.
Another aspect of the present invention is a method for treating a subject infected with HIV-1. The method comprises the steps of:
(1) isolating T cells infected with HIV-1 from the subject;
(2) contacting the infected T cells with a antibody capable of disrupting cell-surface interactions between CD4+ T lymphocytes and monocytes n order to inhibit the production of HIV-1 in the infected T cells, the cells being contacted with a quantity of the antibody sufficient to inhibit the production of HIV-1 in the contacted T cells; and
(3) reinfusing the contacted T cells into the patient to increase the proportion of functional, non-HIV-1-producing T cells in the subject thereby to treat the subject.
In some cases, an additional step of activating the T cells before reinfusion can be desirable.
The same antibodies are preferred for this procedure as for the procedure, described above, of inhibiting HIV-1 production in HIV-1-infected cells. The antibody can also be conjugated to a therapeutic agent. The method can further comprise the step of treating the subject with at least one agent specific for an opportunistic infection associated with AIDS. The agent can be aerosolized pentamidine, isoniazid, rifampin, or amphotericin B.
Reinfusing the contacted T cells into the patient can reduce the complications associated with HIV-1 infection.
Antibodies of the present invention can also be used for in vivo treatment of patients infected with HIV-1. An in vivo treatment procedure comprises administering to a patient whose T cells are infected with HIV-1 a antibody capable of disrupting cell-surface interactions between CD4+ T lymphocytes and monocytes in order to inhibit the production of HIV-1 in the infected T cells, the antibody being administered to the patient in quantity sufficient to inhibit the production of HIV-1 in infected T cells in the patient. The antibody can be conjugated to a therapeutic agent. Treatment can further comprise the step of treating the patient with at least one agent specific for an opportunistic infection associated with AIDS. The antibody can be administered to the patient during viremia.